CN113116885A - Application of tea polyphenol compounds in preparation of antithrombotic drugs - Google Patents

Application of tea polyphenol compounds in preparation of antithrombotic drugs Download PDF

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CN113116885A
CN113116885A CN202110524863.9A CN202110524863A CN113116885A CN 113116885 A CN113116885 A CN 113116885A CN 202110524863 A CN202110524863 A CN 202110524863A CN 113116885 A CN113116885 A CN 113116885A
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morin
silybin
pdi
tea polyphenol
polyphenol compounds
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黄明东
陈丹
袁彩
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Fuzhou University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/357—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having two or more oxygen atoms in the same ring, e.g. crown ethers, guanadrel
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors

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Abstract

The invention discloses application of tea polyphenol compounds in inhibiting protein disulfide isomerase, wherein the tea polyphenol compounds comprise silybin and morin. The invention discovers for the first time that silybin and morin can be combined with Protein Disulfide Isomerase (PDI) and have PDI inhibition activity, so that the silybin and morin can be used for developing and preparing medicines for preventing thrombotic diseases and has wide application prospect.

Description

Application of tea polyphenol compounds in preparation of antithrombotic drugs
Technical Field
The invention relates to the field of biological medicines, in particular to a small molecule inhibitor of silybin and morin as protein disulfide isomerase and application thereof in preparing antithrombotic medicines.
Background
Infectious diseases have been effectively controlled since the twentieth century, the world population has grown enormously, the average human life has generally been prolonged, but on the other hand, non-infectious diseases are becoming the leading cause of human death and disability worldwide. Wherein, cardiovascular and cerebrovascular diseases are the main factors causing the burden of non-infectious diseases, including angina pectoris, myocardial infarction, apoplexy, heart failure, hypertensive heart disease, rheumatic heart disease, myocarditis, thromboembolic diseases, venous thrombosis, etc. Thrombosis is the most common potential cause of three main cardiovascular and cerebrovascular diseases, namely ischemic heart disease (acute coronary syndrome), cerebral apoplexy and venous thromboembolism, and is the leading cause of the death rate of global diseases. Thrombosis mainly occurs in arterial and venous circulation, is a series of pathological changes which are mainly characterized by changing the blood coagulation state and can be divided into arterial thrombosis, venous thrombosis, late stent thrombosis and the like, and local blood flow is blocked or downstream blood flow is blocked by fallen emboli after thrombosis, so that organ tissue ischemia and necrosis are caused.
At present, antithrombotic drugs mainly comprise antiplatelet drugs and anticoagulant drugs. The anticoagulant is mainly used for preventing thrombosis in clinic, such as postoperative deep vein thrombosis, heart and brain thrombosis, pulmonary embolism and the like, and mainly comprises heparins, direct thrombin inhibitors, vitamin K antagonists and FXa factor inhibitors. The general heparin (UFH) can change the configuration of antithrombin III by combining lysine residues in the structure of the antithrombin III, and the allosterized antithrombin III can form a compound with various blood coagulation factors and make the latter lose activity, thereby playing the role of anticoagulation. The direct thrombin inhibitor achieves the anticoagulation purpose by directly inhibiting the thrombin activity. Vitamin K antagonists inhibit the coagulation pathway by competitively inhibiting vitamin K, which is necessary for the modification and activation of coagulation factors. The FXa factor inhibitor achieves the aim of anticoagulation by directly inhibiting the activity of activated coagulation factor X (FXa). However, since thrombin and FXa factor not only participate in thrombosis, but also play a direct and key role in the normal blood coagulation process, the normal blood coagulation function is damaged due to long-term administration of the currently clinically available anticoagulant drugs, and a potential bleeding risk exists. Therefore, there is a need to develop new anticoagulant drugs that do not affect the normal coagulation system and do not risk bleeding while performing antithrombotic therapy.
Protein Disulfide Isomerase (PDI) is one of the members of the thiol isomerase family (TI), and is mainly present in the intracellular reticulum (ER), and plays an essential role in the correct folding of proteins. PDI may function as an oxidoreductase, isomerase, or chaperone. It not only catalyzes the disulfide bond formation of a nascent peptide chain in the endoplasmic reticulum and the rearrangement of mismatched disulfide bonds in molecules, but also can help the nascent protein to be correctly folded, and plays an important role in maintaining the homeostasis of cells. Recent studies have shown that PDI is rapidly secreted from endothelial cells and platelets to the outside during in vivo thrombosis; and in several thrombosis models, inhibition of PDI using neutralizing antibodies can simultaneously inhibit platelet aggregation and fibrin formation at the site of vascular injury, thereby preventing thrombosis. These results indicate that extracellular PDI plays an important role in the initiation of thrombosis. Furthermore, inhibition of PDI has little effect on the normal clotting process (Lin L, Gopal S, Sharda A, Passam F, Bowley SR, Stopa J, Xue G, Yuan C, Furie BC, Flamenhaw R, Huang M, Furie B. Quercetin-3-rutinoside inhibitors Protein variants by bound materials B' x domain. J Biol chem. 2015 Sep 25;290 (23539) JCI, 2012-Jun; 122(6): 13.) the results of the methods of Protein kinase inhibitors inhibition cement a. A. of antithromosomal agents are described in FIGS. Therefore, PDI can be used as a novel antithrombotic therapeutic target.
Disclosure of Invention
The application of tea polyphenol compounds in inhibiting protein disulfide isomerase is disclosed, wherein the tea polyphenol compounds comprise silibinin and morin.
The invention provides application of silybin and morin in preparing antithrombotic medicaments by utilizing the characteristic that silybin and morin have PDI (platelet aggregation inhibitor) inhibitory activity, thereby providing two safe and effective small molecular compounds for clinical antithrombotic treatment.
The tea polyphenol compounds are used for inhibiting the activity of protein disulfide isomerase and inhibiting the platelet aggregation and fibrin generation at the damaged part of blood vessel, thereby preventing thrombosis.
The antithrombotic drug is prepared into tablets, capsules, granules, oral liquid or injections by using silybin, morin or pharmaceutically acceptable salts, hydrates, solvates, crystal forms or diastereoisomers of the molecular structures of the silybin and the morin as active pharmaceutical ingredients.
Silibinin used in the invention has the English name Silibinin and the molecular formula is C25H22O10Molecular weight is 482.44, and its chemical structure is as follows:
Figure DEST_PATH_IMAGE002
。
the Morin used in the invention is called Morin in English, and the molecular formula is C15H10O7Molecular weight is 302.24, and its chemical structure is as follows:
Figure DEST_PATH_IMAGE004
。
in order to achieve the purpose, the invention adopts the following technical scheme:
the application of silybin and morin in preparing antithrombotic drugs utilizes the characteristic that silybin and morin have PDI inhibitory activity, and silybin and morin or pharmaceutically acceptable salts, hydrates, solvates, crystal forms or diastereoisomers thereof are used as pharmaceutically active ingredients to prepare any pharmaceutically acceptable dosage forms, including tablets, capsules, granules, oral liquid or injections.
The IC50 of the in vitro PDI binding assay of silybin and morin are 93.8 μ M and 54.4 μ M respectively; an enzyme inhibition activity experiment shows that compounds with different concentrations have better inhibition activity on PDI; the experimental result of the current-induced carotid thrombosis animal model shows that silybin or morin can obviously improve the thrombosis of mice; the bleeding risk evaluation experiment result shows that the administration of the silibinin or the morin does not prolong the bleeding time of the tail of the mouse and does not influence the whole blood coagulation of the mouse. Therefore, silibinin and morin or pharmaceutically acceptable salts, hydrates, solvates, crystal forms or diastereomers thereof can be used as a PDI inhibitor for preparing antithrombotic drugs.
Due to the application of the technical scheme, the invention has the beneficial effects that:
PDI enzyme plays an important role in the initial stage of thrombosis, can be used as a new target point of antithrombotic treatment, and the inhibition of PDI enzyme does not influence the normal blood coagulation function. The invention discovers that the silybin and the morin have the function of a PDI enzyme inhibitor, can inhibit the generation of thrombus in vivo and do not cause bleeding risk. In vitro experiments prove that silybin and morin can be combined with PDI and inhibit the reduction activity of the PDI, and are inhibitors of the PDI; in vivo experiments prove that the two PDI inhibitors can effectively delay the embolism time of the mouse common carotid artery under direct current stimulation, play a role in resisting thrombus and can not cause bleeding complications. Therefore, the silybin and the morin can be used as inhibitors of PDI and applied to the preparation of antithrombotic therapeutic drugs.
Drawings
FIG. 1 shows the in vitro binding and median Inhibitory Concentration (IC) of silybin to PDI enzyme50）。
FIG. 2 shows the in vitro binding and median Inhibitory Concentration (IC) of morin for PDI enzyme50）。
FIG. 3 is a concentration-dependent inhibition of PDI enzyme activity by silybin.
FIG. 4 is a graph of concentration-dependent inhibition of PDI enzyme activity by morin.
FIG. 5 is a graph comparing silybin and morin to the time of current-induced carotid thrombosis.
FIG. 6 is a graph of the risk of bleeding caused by silibinin and morin.
Detailed Description
For a better understanding of the present invention, reference is made to the following detailed description taken in conjunction with the accompanying drawings and specific examples, but the scope of the invention as claimed should not be limited to the scope of the examples. Unless otherwise indicated, reagents and materials used in the following examples are commercially available. In the following examples, various procedures and methods not described in detail are conventional methods well known in the art.
Example 1: in vitro binding of Silibinin or morin to PDI enzyme
The experimental method comprises the following steps: PDI is generated by experimentsMin unit purification, see documents X Wang, G Xue, M Song, P Xu, D Chen, C Yuan, L Lin, R Flamenhaw, J Li, M Huang (2018), Molecular basis of peptide inhibition of Protein Diagnosis Isomerease (PDI) by combined in silica and experimental methods, RSC Advances 8 (33), 18480 and 18491, purity greater than 95% by SDS-PAGE. 10 microliters of silybin or morin dissolved in DMSO at different concentrations were added to a 96-well plate, 90 microliters of a PDI-containing buffer solution (50 mM Tris-HCl, 150 mM NaCl, pH 7.4) was added to each well, and after incubation at room temperature for 10 minutes, the plate was excited at 278 nm in a microplate reader (Spectra Max i3x, Molecular Devices, U.S.), and emission at 500 nm was detected at 300-. Each test was repeated at least 3 times and averaged. The data processing adopts Graphpad Prism 8 software, and the non-linear fitting is carried out by the fluorescence peak values corresponding to different small molecular compound concentrations to calculate IC50。
The experimental results are as follows:
as shown in FIGS. 1 and 2, the present invention employs a direct binding assay based on intrinsic fluorescence reduction of PDI to determine the binding of small molecule compounds to PDI. When excited at 278 nm, the PDI emits fluorescence at 350 nm, when bound to silybin or morin, the fluorescence decreases and is concentration dependent, and the IC of silybin and morin is obtained by fitting5093 μ M and 54 μ M, respectively. In addition, the emission peak of PDI was red-shifted in combination with silybin, and blue-shifted in combination with morin.
Example 2: silibinin or morin inhibits the reduction activity of PDI enzyme
The experimental method comprises the following steps: PDI reductase activity was determined by the thiol isomerase catalyzing the insulin reduction reaction in the presence of DTT. The aggregation of reduced insulin chains has a significant absorption at 650 nm. The reaction was a buffer solution (50 mM Tris-HCl, 150 mM NaCl, 1 mM DTT, 1 mM EDTA, pH 7.4) containing 500. mu.M insulin, 1.5. mu.M PDI in a total volume of 100. mu.l. 2 microliters of silybin or morin dissolved in DMSO at different concentrations were added to a 96-well plate, 38 microliters of buffer solution and 10 microliters of PDI solution were added to each well, and after incubation at room temperature for 10 minutes, 50 microliters of insulin solution was added and immediately placed in an enzyme-labeling apparatus (Spectra Max i3x, Molecular Devices, U.S.) and the change in absorbance at 650 nm was detected at 37 ℃.
The experimental results are as follows:
as shown in FIGS. 3 and 4, the addition of silybin and morin, respectively, delayed the progress of the reaction in which PDI catalyzes the reduction of insulin by DTT. When the concentration of the silybin and the morin is 100 mu M, the inhibition effect is obvious.
Example 3: effect of Silibinin or morin on Current-induced arterial thrombosis
The experimental method comprises the following steps:
ICR mice were randomly divided into four groups, each group containing 7-9 mice, and were first administered by intraperitoneal injection (vitamin C saline containing 5% DMSO, 20% PEG 300, 2.5% Tween-80, wherein the vitamin C concentration was 2 mg/mL), at a dose of 0.04 mmol/kg, and the saline-administered group and the placebo-administered group were used as controls. After 0.5 hour of dosing, mice were anesthetized (1.5% sodium pentobarbital, 30 mg/kg, i.p.) and left common carotid arteries exposed, stimulated with 0.05 mA of current to disrupt the vessel wall, thereby forming a mixed thrombus in the vessels by a YLS-14B animal thrombometer, recording the rate of occlusion of carotid blood flow every 4 seconds by an infrared detector, and recording the average time to form an occlusive thrombus in the carotid arteries (occlusion rate of 95%).
The experimental results are as follows:
as shown in fig. 5, the carotid artery of the mice in the saline group was blocked within 72 seconds after the electric stimulation, and the carotid artery of the mice in the placebo group was blocked within 73 seconds after the electric stimulation, and the two groups were compared without significant difference. The 0.04 mmol/kg silibinin and morin group respectively prolongs the blood vessel blockage time to 154 seconds and 198 seconds, and the difference is significant compared with the normal saline group (pValues of 0.017 and 0.001, respectively). The experiment shows that the silybin and the morin can effectively prolong the thrombus formation time in the carotid artery thrombus electrically stimulated model and have an anticoagulation effect.
Example 4: evaluation of the risk of bleeding caused by Silibinin and morin by tailgating experiments
The experimental method comprises the following steps:
the group administration was the same as in example 3, in which ICR mice were randomly divided into four groups, each group containing 6 mice, and was first administered by intraperitoneal injection (vitamin C physiological saline containing 5% DMSO, 20% PEG 300, 2.5% Tween-80 in which the vitamin C concentration was 2 mg/mL) at a dose of 0.04 mmol/kg, and the physiological saline-administered group was used as a control. After 0.5 hour of administration, the mice were anesthetized (1.5% sodium pentobarbital, 30 mg/kg, i.p.), and the tail of the mice was cut off 10 mm from the tip, immediately immersed in 10 mL of isotonic saline at 37 ℃, and the length of bleeding was recorded after stopping bleeding. The amount of blood lost was quantified by measuring the amount of hemoglobin collected in 10 mL of isotonic saline, collecting the red blood cells after centrifugation at 1500 rpm, and lysing with 2 mL of lysis buffer (8.3 g/L NH)4Cl, 1 g/L KHCO30.037 g/L EDTA) and finally the absorbance at 570 nm of each sample was determined by a microplate reader.
The experimental results are as follows:
as shown in FIG. 6, the bleeding time and amount were not significantly increased in the 0.04 mmol/kg silibinin and morin group compared to the saline group, and there was no significant difference compared to the saline group. The results of this experiment show that administration of silibinin and morin does not create a bleeding risk.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.

Claims (3)

1. The application of tea polyphenol compounds in inhibiting protein disulfide isomerase is characterized in that the tea polyphenol compounds comprise silybin and morin.
2. An antithrombotic agent characterized by inhibiting protein disulfide isomerase activity and inhibiting platelet aggregation and fibrin formation at a site of vascular injury by using the tea polyphenol compound of claim 1, thereby preventing thrombosis.
3. The drug according to claim 2, wherein the antithrombotic drug is a tablet, capsule, granule, oral liquid or injection prepared from silybin, morin or a pharmaceutically acceptable salt, hydrate, solvate, crystal form or diastereomer of the molecular structure thereof as a pharmaceutically active ingredient.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114272379A (en) * 2021-11-26 2022-04-05 中国科学院生物物理研究所 Novel small molecule inhibitor targeting Ero1 alpha/PDI electron transfer system

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101062030A (en) * 2006-04-26 2007-10-31 哈药集团中药二厂 Method of preparing silybin frozen powder injection
CN105311012A (en) * 2014-06-24 2016-02-10 上海中医药大学 New medicinal application of morin hydrate

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101062030A (en) * 2006-04-26 2007-10-31 哈药集团中药二厂 Method of preparing silybin frozen powder injection
CN105311012A (en) * 2014-06-24 2016-02-10 上海中医药大学 New medicinal application of morin hydrate

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CHIH-WEI HSIA等: "Novel Therapeutic Agent against Platelet Activation In Vitro and Arterial Thrombosis In Vivo by Morin Hydrate", 《INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES》 *
MICHAL BIJAK等: "Flavonolignans reduce the response of blood platelet to collagen", 《INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES》 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114272379A (en) * 2021-11-26 2022-04-05 中国科学院生物物理研究所 Novel small molecule inhibitor targeting Ero1 alpha/PDI electron transfer system

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